In gene therapy and many other therapeutically relevant biochemical and biotechnological applications the use of nucleic acids for therapeutic and diagnostic purposes is of major importance. As an example, rapid progress has occurred in recent years in the field of gene therapy and promising results have been achieved. Nucleic acids are therefore regarded as important tools for gene therapy and prophylactic and therapeutic vaccination against infectious and malignant diseases.
Nucleic acids, both DNA and RNA, have been used widely in gene therapy, either in naked or in complexed form. In this context, the application of nucleic acids and particularly of RNA for therapeutic vaccination is revised permanently. On the one hand, nucleic acids and particularly RNA or mRNA molecules can be optimized for a more efficient transcription rate. The 5′ Cap structure, the untranslated and translated regions are typically modified to stabilize the molecule or to change its characteristics to enhance its translation properties (see e.g. Pascolo, S. (2008), Handb Exp Pharmacol (183): 221-35). Further, different formulations of nucleic acids and particularly of mRNA molecules or different delivery routes are investigated to achieve improved expression levels. To mention are the encapsulation into cationic liposomes or cationic polymers (see e.g. Hoerr, I., R. Obst, et al. (2000), Eur J Immunol 30(1): 1-7.; Hess, P. R., D. Boczkowski, et al. (2006), Cancer Immunol Immunother 55(6): 672-83; Scheel, B., R. Teufel, et al. (2005), Eur J Immunol 35(5): 1557-66), the needleless delivery of gold particles coated by mRNA using a gene gun (see e.g. Qiu, P., P. Ziegelhoffer, et al. (1996). “Gene gun delivery of mRNA in situ results in efficient transgene expression and genetic immunization.” Gene Ther 3(3): 262-8), the transfection of in vitro generated autologous APCs that are re-administred to patients (see e.g. Boczkowski, D., S. K. Nair, et al. (1996). “Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.” J Exp Med 184(2): 465-72; Boczkowski, et al, 1996, supra), and the direct injection of naked RNA (see Hoerrr et al, 2000, supra). Despite all progress achieved regarding gene delivery it is very important to improve further transfection efficiency to make nucleic acids especially RNA applicable for all imaginable therapeutic purposes.
Application of RNA thus represents a favored tool in modern molecular medicine. It also exhibits some superior properties over DNA cell transfection. As generally known, transfection of DNA molecules may lead to serious problems. E.g. application of DNA molecules bears the risk that the DNA integrates into the host genome. Integration of foreign DNA into the host genome can have an influence on expression of the host genes and possibly triggers expression of an oncogene or destruction of a tumor suppressor gene. Furthermore, a gene—and therefore the gene product—which is essential to the host may also be inactivated by integration of the foreign DNA into the coding region of this gene. There may be a particular danger if integration of the DNA takes place into a gene which is involved in regulation of cell growth. Nevertheless, DNA still represents an important tool, even though some risks are associated with the application of DNA. These risks do not occur if RNA, particularly mRNA, is used instead of DNA. An advantage of using RNA rather than DNA is that no virus-derived promoter element has to be administered in vivo and no integration into the genome may occur. Furthermore, the RNA has not to overcome the barrier to the nucleus. However, a main disadvantage resulting from the use of RNA is due to its huge instability. Even though it is understood that DNA, e.g., naked DNA, introduced into a patient' circulatory system is typically not stable and therefore may have little chance of affecting most disease processes (see e.g. Poxon et al., Pharmaceutical development and Technology, 5(1), 115-122 (2000)) the problem of stability is even more evident in the case of RNA. As generally known, the physico chemical stability of RNAs in solution is extremely low. RNA is very susceptible to hydrolysis by ubiquitous ribonucleases and is typically completely degraded already after a few hours or days in solution. This even occurs in the absence of RNases, e.g. when stored a few hours or days in solution at room temperature.
To avoid such degradation the RNA is typically stored at −20° C. or even −80° C. and RNAse free conditions to prevent a prior degradation of the RNA. This method, however, does not prevent a loss of function effectively and additionally is very cost-intensive for shipping when these temperatures have to be guaranteed. One further method for stabilization comprises lyophilization or freeze-drying of the RNA. Lyophilization is a worldwide known and recognized method in the art to enhance storage stability of temperature sensitive biomolecules, such as nucleic acids. During lyophilization, typically water is removed from a frozen sample containing nucleic acids via sublimation. The process of lyophilization is usually characterized by a primary and a secondary drying step. During the primary drying step, free, i.e. unbound, water surrounding the nucleic acid (sequence) and optionally further components, escapes from the solution. Subsequent thereto water being bound on a molecular basis by the nucleic acids may be removed in a secondary drying step by adding thermal energy. In both cases the hydration sphere around the nucleic acids is lost.
During lyophilization the sample containing nucleic acids is initially cooled below the freezing point of the solution and accordingly of the water contained therein. As a result, the water freezes. Dependent on temperature, rate of cooling down (freezing rate), and the time for freezing, the crystal structure of water is changed. This exhibits physical stress on the nucleic acid (sequence) and other components of the solution, which may lead to a damage of the nucleic acid, e.g. breakage of strands, loss of supercoiling, etc. Furthermore, due to the decrease of volume and loss of the hydration sphere, autocatalytic degradation processes are favored e.g. by traces of transition metals. Additionally, significant changes of pH are possible by concentration of traces of acids and bases.
Lyophilization involves two stresses, freezing and drying. Both are known to damage nucleic acids, such as non-viral vectors or plasmid DNA. In the literature, a number of cryoprotectants and lyoprotectants are discussed for lyophilization purposes to prevent these damages. In this context, cryoprotectants are understood as excipients, which allow influencing the structure of the ice and/or the eutectical temperature of the mixture. Lyoprotectants are typically excipients, which partially or totally replace the hydration sphere around a molecule and thus prevent catalytic and hydrolytic processes.
In the specific context of DNA, lyophilization causes the removal of the hydration sphere around the DNA, wherein it appears that there are approximately 20 water molecules per nucleotide pair bound most tightly to DNA. These water molecules do not form an ice-like structure upon low-temperature cooling. Upon DNA dehydration over hygroscopic salts at 0% relative humidity, only five or six water molecules remain (see e.g. Tao et al., Biopolymers, 28, 1019-1030 (1989)). Lyophilization may increase the stability of DNA under long-term storage, but may also cause some damage upon the initial lyophilization process, potentially through changes in the DNA secondary structure, breaks of the nucleic acid chain(s) or the concentration of reactive elements such as contaminating metals. Lyophilization can also cause damage upon the initial lyophilization process in other nucleic acid, e.g. RNA. Agents that can substitute for non-freezable water, such as some carbohydrates, can demonstrate cryoprotective properties for DNA and other molecules during lyophilization of intact bacteria (see e.g. Israeli et al, Cryobiology, 30, 519-523 (1993); or Rudolph et al, Arch. Biochem. Biophys., 245, 134-143 (1986)).
During lyophilization, specific carbohydrates, such as several sugars, appear to play a central role in stabilization of the nucleic acid. However, when using cryoprotectants and lyoprotectants, no general rule may be applied with respect to their impact on different groups of compounds. Therefore, in many cases an optimal formulation has to be found using empirical methods.
In this context, specific carbohydrates are utilized in the art as lyoprotective substances for enhancing stability of the nucleic acid (sequence) during lyophilization. They exhibit an effect on storage stability after lyophilisation of pure nucleic acids or nucleic acid (sequence) complexes (see e.g. Maitani, Y., Y. Aso, et al. (2008), Int J Pharm 356(1-2): 69-75; Quaak, S. G., J. H. van den Berg, et al. (2008), Eur J Pharm Biopharm 70(2): 429-38; Jones, K. L., D. Drane, et al. (2007), Biotechniques 43(5): 675-81; Molina, M. C., S. D. Allison, et al. (2001), J Pharm Sci 90(10): 1445-55; and Allison, S. D. and T. J. Anchordoquy (2000), J Pharm Sci 89(5): 682-91). Lyoprotective properties are particularly described for sucrose, glucose, and trehalose. They allow to restore at least in part the transfection efficiency which is otherwise lost in many cases after lyophilisation (see Maitani et al, 2008, supra; Yadava, P., M. Gibbs, et al. (2008). AAPS PharmSciTech 9(2): 335-41; Werth, S., B. Urban-Klein, et al. (2006), J Control Release 112(2): 257-70; Brus, C., E. Kleemann, et al. (2004), J Control Release 95(1): 119-31; Poxon, S. W. and J. A. Hughes (2000), Pharm Dev Technol 5(1): 115-22; Anchordoquy, T. J., J. F. Carpenter, et al. (1997), Arch Biochem Biophys 348(1): 199-206). Sugars are able to prevent loss in activity due to the lyophilization process mainly by preventing particle fusion/aggregation especially in the case of liposome complexed nucleic acids (see Yadava et al, 2008, supra; Katas, H., S. Chen, et al. (2008), J Microencapsul: 1-8; Molina et al, supra, 2001).
Particularly, Poxon et al. (2000, supra) investigated the effect of lyophilization on plasmid DNA activity. Poxon et al. (2000, supra) hypothesized, that a change in the DNA conformation from supercoiled to open circular and linear form would be indicative of damage of the plasmid DNA. However, the percentage of supercoiled DNA did not change after lyophilization and subsequent DMED treatment, suggesting that other effects drew responsible for the loss of transfection efficiency. Poxon et al. (2000, supra) found that a decrease in plasmid DNA activity as measured by an in vitro transfection assay can be ameliorated by the use of carbohydrates during lyophilization of the plasmid DNA but he did not found that any of the used carbohydrates increased the transfection efficiency of the plasmid DNA. As lyoprotectants, glucose (monosaccaride), sucrose and lactose (disaccharides) were used. Poxon et al. (2000, supra), however, only carried out investigations with plasmid DNA. They did also not investigate if the addition of saccharides to the lyophilization affects the stability of the lyophilized plasmid DNA.
Li et al. (see Li, B., S. Li, et al. (2000), J Pharm Sci 89(3): 355-64) furthermore showed that disaccharides are superior to monosaccharides using them as a cryoprotectant for lyophilization of lipid based gene delivery systems due to the prevention of aggregation. They noted that it is very important to prevent the particle size of the complexes during lyophilization. Unfortunately, in a specific example of lipid based gene delivery systems, lyophilization with mannose led to an increase in particle size, which was regarded as negative for transfection efficiency. Additionally Li et al. (2000, supra) showed that lipid delivery systems can be stored at room temperature without loss of transfection efficiency when lyophilized in 10% sucrose. Li et al. (2000, supra) did not examine the stabilization due to the presence of mannose as a lyoprotectant. More importantly, they did not observe an increase in the expression of the encoded protein due to the presence of sugar (sucrose and trehalose) in the injection buffer.
Even though many available prior art documents discuss the stabilization of nucleic acids during lyophilization in the context of plasmid DNA, only few publications focus on stabilization of other nucleic acids, such as RNAs, e.g. during lyophilization and long-term storage.
In this context, Jones et al (2007, supra) is one rare document, which examines the effect of sugars on long term stability of mRNA. It describes the possibility to prevent storage depending loss of transfection activity in vitro. Jones et al (2007, supra) uses trehalose as a lyoprotectant and shows a preventive effect on the loss of transfection activity at a storage temperature of 4° C. for a period of 6 months. Integrity of the mRNA was only measured by loss of weight after recovering. At elevated temperatures (room temperature and higher) degradation and a dramatic loss of transfection efficiency took place. Additionally; transfection efficiency could not be improved using trehalose as lyoprotectant.
In a further context, specific carbohydrates may also be utilized to improve biological activity and/or transfection efficiency, which is, at least at a first glance, independent from stability issues. Such an effect of specific carbohydrates, e.g. of mannose may be attributed to the interaction of these carbohydrates with specific receptors in the cell. As an example, the addition of mannose may involve the mannose receptor targeted transfer. The mannose receptor (MR) is primarily present on dendritic cells (DCs) and macrophages. The carbohydrate recognition domains of the MR recognizes carbohydrates (e.g. mannose, fucose, glucose, N-Acetylglucosamine, maltose) on the cell walls of infectious agents (mainly bacteria and yeast) which leads to rapid internalization and phagocytosis. This process can initiate effective immune defense. Several different strategies targeted to the MR have been used to enhance transfection levels or to develop upgraded vaccines (see Keler, T., V. Ramakrishna, et al. (2004). “Mannose receptor-targeted vaccines.” Expert Opin Biol Ther 4(12): 1953-62). In this context, mannose modified non-viral DNA vectors, including cationic liposomes (Kawakami, S., A. Sato, et al. (2000), Gene Ther 7(4): 292-9; and Hattori, Y., S. Kawakami, et al. (2006), J Gene Med 8(7): 824-34), polyethyleneimine (Diebold, S. S., H. Lehrmann, et al. (1999), Hum Gene Ther 10(5): 775-86), poly L-lysine (Nishikawa, M., S. Takemura, et al. (2000), J Drug Target 8(1): 29-38), dendrimers (Arima, H., Y. Chihara, et al. (2006), J Control Release 116(1): 64-74) and chitosan (Kim, T. H., J. W. Nah, et al. (2006), J Nanosci Nanotechnol 6(9-10): 2796-803); (Hashimoto, M., M. Morimoto, et al. (2006), Biotechnol Lett 28(11): 815-21), have been reported (see also review: Irache, J. M., H. H. Salman, et al. (2008). “Mannose-targeted systems for the delivery of therapeutics.” Expert Opin Drug Deliv 5(6): 703-24). However, in all cases mannose was covalently bound to the vector to ensure a combined uptake due to binding to the mannose receptor. However, the expression of the mannose receptor is restricted to a few cell types (especially dendritic cells) which are not excessively present in the dermis and therefore it appeared unlikely that free mannose improves the expression of the encoded protein due to an increased uptake in mannose receptor expressing cells.
The only case which is known in the prior art to use free sugar to enhance transfection efficiency is disclosed in Sun et al (see Sun, C., K. Ridderstrale, et al. (2007), Plant J 52(6): 1192-8). Sun et al. (2007) could show that sucrose can stimulate uptake of oligo deoxynucleotides (ODN) in human cells (in vitro). They investigated the effect of glucose and sucrose to the ODN delivery compared to the effect of oligofectamine, a commercially available lipid-based transfection reagent. Interestingly they observed that sucrose was 30% more potent than oligofectamine and even 60% more potent than glucose supporting ODN uptake. They hypothesized that sucrose is a common trigger for endocytosis in animal cells and therefore the ODN internalizes into endosomes together with the sucrose. Sun et al. (2007) only examined in vitro transfection assays which are very difficult to transfer to the in vivo situation due to the dilution effect. In tissues it thus appeared very unlikely that the nucleic acid and the sugar molecule enter the cell at the same time.
Summarizing the above, there is a long-lasting and urgent need in the art to provide means, which allow (a skilled person) to store RNA without a loss in activity, an effect, which is observed in many cases. Likewise, there is a long-lasting and urgent need to provide means, which allow (a skilled person) to enhance transfection efficiency of nucleic acids especially of RNA for in vitro and particularly for in vivo applications. In this context, a still most challenging problem of the prior art is the stability of the above defined nucleic acids, particularly during storage and delivery. Another challenging problem of the prior art, which in part due to the problem of stability, is the loss of activity subsequent to storage, or the loss of biological activity after lyophilization (e.g. increase in particle size, . . . ), which is observed for many nucleic acids. Finally, a further challenging problem of the prior art represents the small amount of expressed protein or small biological activity of the nucleic acid obtained upon transfection into the cell. Some further problems can be regarded in the provision of a suitable final dosage form for delivering these nucleic acids but also the production, transport and storage thereof. Especially transport of RNA is a remaining problem because it is very cost-intensive to ensure temperatures at −20° C. and below during shipment.